Assembly of the bacterial flagellum requires the action of a specialized secretion apparatus at the base, which transports the needed proteins into a central channel in the structure that conducts them to their site of incorporation at the tip. The flagellar secretion machinery is closely related to the "injectisome" used by many gram-negative pathogens to inject effector proteins into host cells. In both the flagellar and nonflagellar systems this export mechanism is referred to as "type III" secretion. In type III systems gene regulation is coordinated with assembly. How is gene regulation coupled to assembly of large organelles such as the flagellum and the injectasome? Much information has been obtained about how transcription of genes in flagellar and virulence type III systems is coupled to assembly. Little is know about how translation is coupled to assembly of these structures. This proposal will examine the translational control of the flagellar filament protein. It is reported that secretion through the flagellar and injectisome structures can occur through mRNA signals. Addition of the 5'and 3'mRNA untranslated regions of the fliC flagellin is reported to target proteins for secretion through the flagellum. Data suggests that translation of the fliC gene is localized to the base of each flagellum. This would provide a mechanism to independently regulate gene expression for different flagella within an individual cell. This process will be a focus of the studies in this proposal. PUBLIC HEALTH RELEVANCE: A goal of my lab is to understand how flagellar gene expression is coupled to assembly. While much is known about how genes respond to levels of small metabolites, the construction of large organelles poses unique challenges and has offered unique solutions to the coordinated regulation of genes in building complex structures, such as the bacterial flagellum. We know a fair amount about how transcription of flagellar genes is coupled to assembly through the action of a transcriptional inhibitor (FlgM) that is removed at a particular point in flagellar assembly (secretion through a completed hook-basal body structure). A few years ago we observed that increased transcription of the fliC flagellin gene alone did not lead to increased flagellin protein, but the Kutsukake and Yamamoto labs reported that increased transcription of the entire flagellar regulon resulted in a 2- to 4-fold increase in the number of flagellar per cell [1]. This suggested that a mechanism inhibits flagellin post-transcription when it is not coupled to the rest of flagellar biosynthesis. More recent and intriguing work from the Westerlund-Wickstrvm lab reports that any heterologous protein can be secreted through the bacterial flagellum if its corresponding open reading frame is flanked by the 5'and 3'-untranslated regions from the E. coli fliC gene [2]. If true, this is remarkable because it suggests that a peptide secretion signal is not required for secretion through the flagellum and information in the untranslated RNA can target proteins for secretion. Whether secretion is directed by peptide or RNA signals is a long-standing area of controversy in the type III secretion field. Little has been done to characterize the role of translational control in coupling flagellar gene expression to assembly. We propose to address this level of control in this grant application.